Studies on expression of aldehyde dehydrogenase in normal and cancerous tissues of thyroids.

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells.

Orthotopic neobladder reconstruction in elderly bladder cancer patients.

BACKGROUND: We compared the clinical results of orthotopic neobladder reconstruction in elderly patients and those in younger patients retrospectively in order to verify whether age is a critical factor in selecting a method of urinary diversion. METHODS: Following radical cystectomy for bladder cancer, 12 patients aged 75 or older and 17 patients under 75 who underwent orthotopic neobladder reconstruction between January 1992 and May 1999 were investigated in this study. The authors TS and BS were among the surgeons who performed operations for all cases. Of the 12 elderly patients, orthotopic neobladders were constructed according to Hautmann's method in nine cases, Studer's method in one case and Reddy's method in two cases. Of the 17 younger patients, these methods were employed in 12, one and four cases, respectively. Operative procedure, early and late complications, prognosis, continence and voiding pattern were investigated in these patients. RESULTS: The follow-up periods for elderly and younger groups ranged from 21.3 to 82.7 months and from 8.8 to 94.2 months, respectively. No difference in operation time, amount of bleeding or postoperative length of hospitalization was observed between elderly and younger patients. The rates of early complications in elderly and younger patients were 41.7% and 35.3%, respectively. Late complication rates were 33.3% and 47.1%, respectively. The difference in these complication rates was not statistically significant. One of the elderly and two of the younger patients had local recurrence and metastasis postoperatively. Those three patients had died of their bladder cancer. No statistically significant difference between groups was recognized in either cause-specific survival or overall survival, nor was there such a difference in relation to micturition/continence. CONCLUSION: Based on these results, we believe that because age is not a critical factor in the selection of urinary diversion method, neobladder reconstruction following cystectomy for bladder cancer is indicated in elderly patients. As stoma management is difficult for the patients, we consider orthotopic neobladder reconstruction to be the method of choice if the patients' general physical condition allows.

Differentiation stages of eosinophils characterized by hyaluronic acid binding via CD44 and responsiveness to stimuli.

To characterize interleukin (IL)-5-induced eosinophils, we examined the expression of CD44, very late antigen (VLA)-4, and the IL-5 receptor alpha chain, as well as the levels of eosinophil peroxidase and the generation of superoxide. Eosinophils were prepared from IL-5-transgenic mice, then characterized using electron microscopy to determine their responses to stimuli. Whereas CD44 densities remained almost constant, the level of VLA-4 increased in parallel with eosinophil maturation. Although a subset of IL-5-induced eosinophils with high side scatter recovered from bone marrow and rare ones found in blood recognized hyaluronic acid (HA), most did not have this property. Bone marrow eosinophils with high side scatter and lower density contained eosinophil peroxidase, not only in granules, but also in membranous structures for 30% of this population. This population developed HA-binding ability in response to IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1, eotaxin, nerve growth factor (NGF), and opsonized zymosan (OZ). Peripheral blood eosinophils acquired HA-binding ability in response to the same stimuli, but their responses were less than those of bone marrow eosinophils with high levels of side scatter. However, splenic eosinophils did not respond to these stimuli. Although peripheral blood eosinophils did not proliferate when stimulated by IL-5, these were the only cells that released eosinophil peroxidase in response to IL-4, MIP-2, MCP-1, eotaxin, NGF, and OZ. With the exception of a subset of bone marrow eosinophils, the ability to acquire HA binding, but not the ability to generate superoxide, correlated with eosinophil peroxidase activity and major basic protein accumulation in the granules of maturing cells.

Ny-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade.

NY-ESO-1 mRNA expression in transitional cell carcinoma was investigated by reverse transcription-PCR and immunohistochemistry. NY-ESO-1 mRNA was detected in 20 of 62 (32%) tumor specimens. There was a correlation between NY-ESO-1 expression and tumor grade: 0 of 4 (0%) grade 1 (G1), 6 of 26 (23%) grade 2 (G2), and 14 of 32 (44%) grade 3 (G3) tumors were NY-ESO-1 mRNA positive. Immunohistochemical analysis using NY-ESO-1-specific monoclonal antibody ES121 showed that 2 of 14 NY-ESO-1 mRNA-expressing G3 tumors were positive for NY-ESO-1. No NY-ESO-1 staining was observed in the panel of 30 G1 or G2 tumor specimens, including 6 NY-ESO-1 mRNA-positive cases. Sera from an expanded panel of 124 patients with transitional cell carcinoma were tested for the presence of NY-ESO-1 antibody. Seropositivity was observed in 9 of 72 (12.5%) patients with G3 tumors, whereas none of 52 patients with G1 or G2 tumors produced antibody against NY-ESO-1. In the 9 positive patients with NY-ESO-1 antibody, 4 had muscular invasive tumors, and 5 had carcinoma in situ.

Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen.

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

A boy with normal growth in spite of growth hormone deficiency after resection of a suprasellar teratoma.

We reported a boy with panhypopituitarism after removal of a suprasellar teratoma and pituitary stalk transection at the age of 3 months. His growth was accelerated after 5 years of age without growth hormone (GH) therapy, although he had poor height growth until age 4 under treatment with hydrocortisone, levothyroxine sodium, and desamino-D-arginine vasopressin (DDAVP). Hyperphagia and obesity developed after surgery. Endocrinological examination revealed no GH response to glucagon, low serum levels of insulin-like growth factor (IGF)-1 and IGF binding protein-3 (IGFBP-3). Serum prolactin was normal, but serum insulin was high. Some patients who received an operation for craniopharyngioma were reported to achieve normal growth without GH secretion, but the mechanism is still unknown. High serum levels of prolactin or insulin can be associated with normal IGF in GH deficient patients. This patient had obesity and high serum insulin, which may be related to growth without GH secretion.

A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils.

In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.

Polymyositis associated with primary cytomegalovirus infection.

A case of polymyositis associated with primary cytomegalovirus infection in a 17-y-old girl is reported. The girl exhibited fever, sore throat, progressive myalgia and muscle weakness with elevated creatine kinase, atypical lymphocytosis and myopathic features in the electromyogram. Histopathologically, biopsied muscle met the criteria for polymyositis. Primary cytomegalovirus infection was proven by seroconversion of IgG as well as IgM antibodies. This is the first report of an association between cytomegalovirus infection and polymyositis.

A variant of myelokathexis with hypogammaglobulinemia: lymphocytes as well as neutrophils may reverse in response to infections.

A 7-year-old boy with prolonged and marked leukopenia diagnosed at 6 months of age is described. The polymorphonuclear cells presented no hypersegmented nuclei or concentrated nuclear chromatin, although vacuolated myeloid cells appeared in bone marrow smears. Neutrophils reversed in response to administration of G-CSF. His leukocyte counts were 400-1000/microL during afebrile periods and increased to 2000-3000/microL in response to infections. The increased leukocyte was usually neutrophils, but lymphocytes also increased at EB-virus infection. The serum IgG decreased gradually and was 364 mg/dL at 7 years of age. Antibody responses were normal and recurrent otitis media has been the patient's only problem. Granulocytopenia with hypogammaglobulinemia of this patient mimics myelokathexis with hypogammaglobulinemia, and lymphocytes also increased at viral infections.

Functional expression of transmembrane 4 superfamily molecules on human eosinophils.

BACKGROUND: Transmembrane 4 superfamily (TM4SF) molecules are exclusively found on hematopoietic cells. Several members of the TM4SF are reported to be associated with other cell surface molecules, including integrins, and might participate in signal transduction, but little is known about their role on eosinophils. In the present study, we determined the expression and function of TM4SF molecules on human eosinophils. METHODS: Surface expression of TM4SF molecules on purified peripheral blood eosinophils was examined using indirect immunofluorescence and flow cytometry. Purified eosinophils were incubated with anti-TM4SF monoclonal antibodies (mAbs) for up to 24 h. Eosinophil activation was evaluated by measuring eosinophil homotypic aggregation as well as changes in surface expression of CD11b or CD62L by flow cytometry. RESULTS: Freshly isolated eosinophils expressed CD9, CD37, CD53, CD63 and CD81. Incubation with anti-CD9 mAb but not with anti-CD37, CD53, CD63 or CD81 mAb induced significant eosinophil homotypic aggregation. Incubation with any of the anti-TM4SF mAb for 30 min failed to alter the expression of either CD11b or CD62L on eosinophils. In contrast, the expression of CD11b was significantly enhanced after 24 h of incubation with anti-CD53 mAb, while the expression of CD62L was significantly reduced with anti-CD81 mAb. CONCLUSIONS: Cross-linking of some surface TM4SF molecules induced significant eosinophil homotypic aggregation, upregulation of CD11b expression, or CD62L shedding, consistent with activation of eosinophils. Our data suggest that several TM4SF molecules are functionally expressed on human eosinophils, and therefore might participate in allergic inflammation.

CD34 expression as a novel marker of transformed mesangial cells in biopsied glomerular diseases.

CD34 is a marker of haematopoietic progenitor cells, stromal precursors, vascular endothelial cells, and a variety of stromal tumour cells. This immunohistochemical study examined the CD34 expression of glomerular mesangial cells in normal and diseased glomeruli and compared it with the staining patterns of alpha-smooth muscle actin (ASMA), as a transformed mesangial cell marker, and CD31, as an endothelial cell marker. In addition, the CD34 and ASMA expression of mesangial cells in various glomerulonephritis and the relationship of the immunostaining intensity to the severity of IgA nephropathy were semiquantitatively evaluated. In normal glomeruli, all cell types were negative for CD34, but in glomeruli in mesangial proliferative glomerulonephritis, CD34 was expressed exclusively in mesangial cells, corresponding to ASMA expression. The dendritic and scattered staining pattern, the mesangial location of positive signals, and the enhanced expression were clearly different from CD31 expression in diseased glomeruli. In comparison with normal controls, the grade of immunostaining for CD34 (CD34 INDEX) in mesangial proliferative glomerular diseases was higher than that of ASMA (ASMA INDEX). With the severity of glomerulonephritis, the CD34 INDEX gradually increased. These studies indicate that CD34 is a useful marker of mesangial transformation and that immunohistochemical examination with the anti-CD34 antibody is useful for the diagnosis and stage determination of glomerular diseases.

Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a severe illness with unusual EBV activation that persists for years, and its pathogenesis is largely unknown. After the creation of an accurate and reproducible polymerase chain reaction system to quantify EBV DNA, virus loads in peripheral blood lymphocytes (PBL) were determined in 54 children: 15 with CAEBV, 16 with infectious mononucleosis (IM), and 23 healthy children. Children with CAEBV and those with IM had high virus loads. Lower loads were detected in 47% of seropositive healthy donors. There were two distinct differences between children with CAEBV and those with IM: The former had greater viral replication (10(3)-10(7) copies/2.5x10(5) PBL) than those with IM, and viral replication declined in children with IM whereas active replication persisted for years in subjects with CAEBV. Persisting high virus loads are a possible diagnostic criterion for CAEBV. EBV loads may enable classification and prognosis of EBV infections.

Increased cell-free viral DNA in fatal cases of chronic active Epstein-Barr virus infection.

We have studied the nature of Epstein-Barr virus (EBV) infection in 33 patients with chronic active EBV infection. The study population included 14 patients with fatal chronic EBV infection and 19 patients with nonfatal chronic EBV infection, as well as 18 patients with acute EBV-induced infectious mononucleosis and 10 healthy controls. EBV DNA was measured in serum or plasma samples from the patients by semiquantitative polymerase chain reaction-based assay. EBV DNA was detected in serum or plasma samples from 62% (9/14) of patients with fatal chronic active EBV infection. In contrast, only 11% (2/19) of patients with nonfatal chronic active EBV infection and 11% (2/18) of patients with infectious mononucleosis displayed EBV DNA. None of the healthy controls tested positive. Cell-free circulating EBV DNA may represent an important feature of chronic active EBV infection and may provide a useful tool to monitor the severity of this illness.

Lysosomal glycogen storage disease with normal acid maltase with early fatal outcome.

In a male infant who had cardiomyopathy, generalized muscle weakness and increased serum creatine kinase levels, his muscle biopsy revealed myopathic changes with tiny intracytoplasmic vacuoles containing PAS-positive material and high acid phosphatase activity, but had normal acid maltase activity biochemically. These findings were consistent with those seen in lysosomal glycogen storage disease with normal acid maltase (Danon disease). Sarcolemmal indentations commonly seen in this disease were missing, but a complement membrane attack complex, C5b-9 was positive along the surface membrane of the muscle fibers as seen in X-linked vacuolar myopathy. The patient was on a respirator and died at 27 months of age from pneumonia and hypertrophic cardiomyopathy. Lysosomal glycogen storage disease with normal acid maltase may be manifested at birth with marked skeletal and cardiac involvement leading to death in early infancy.

Combination chemotherapy (cisplatin, vinblastin) and low-dose irradiation in the treatment of pineal parenchymal cell tumors.

Pineal parenchymal cell tumors (PPCTs) with or without metastasis into the lumbar region by way of the cerebrospinal fluid were treated successfully with combination chemotherapy using cisplatin, vinblastin, and bleomycin (PVB) or cisplatin and vinblastin (PV) and low-dose irradiation (25 approximately 30 Gy). Our series included a case of pineoblastoma, two cases of mixed pinocytoma/pineoblastoma, and a case of pineocytoma, compared to which the data held by the All Japan Brain Tumor Registry (AJBTR) included information on 47 cases pineocytoma and 20 of pineoblastoma. All our patients have survived, with scores of 90% or over on Karnofsky's performance scale, for 2-12 years of follow-up so far; however, the 5-year survival rates of the patients recorded by AJBTR were 83% for pineocytoma treated with radiation and 43% without radiation; and 42% for pineoblastoma treated with radiation and 50% without radiation. Incomplete or varied chemotherapeutic regimens used in different medical centers to treat PPCTs precluded an evaluation such as was made by AJBTR. Our results suggested that combination chemotherapy with low-dose back-up radiotherapy may be the treatment of choice for primary or recurrent disease with or without dissemination in PPCTs.

Altered expression of CD11b and CD62L after cross-linking of CD45 isoforms on human eosinophils.

BACKGROUND: Phosphotyrosine phosphatase CD45 is exclusively found on nucleated hematopoietic cells. CD45 is an essential component of signaling of mast cel degranulation and of lymphocyte activation, but little is known about its role in eosinophils. In the present study, we have determined the expression and function of CD45 isoforms on human eosinophils. METHODS: Expression of CD45 isoforms on purified peripheral blood eosinophils was examined using indirect immunofluorescence and flow cytometry. Eosinophils were cultured with anti-CD45 isoform monoclonal antibodies (mAbs) for up to 24 h. Eosinophil activation was evaluated by measuring surface expression of CD11b or CD62L by flow cytometry. RESULTS: Fresh eosinophils express CD45, CD45RB, CD45RO epitopes but not CD45RA. Incubation with anti-CD45 isoform mAb for 30 min did not alter the expression of either CD11b or CD62L on eosinophils. In contrast, the expression of CD11b was significantly enhanced after 24 h of incubation with mAbs against CD45, CD45RB, or CD45RO. In addition, the expression of CD62L was also significantly reduced with anti-CD45RB and with anti-CD45RO mAbs. CONCLUSIONS: Cross-linking of surface CD45 isoforms for 24 h significantly induced upregulation of CD11b expression and CD62L shedding, consistent with activation of eosinophils. Our data suggest that CD45 isoforms are functionally expressed on human eosinophils, and are capable of modulating eosinophil function and might participate in allergic inflammation.

Serodiagnosis of infectious mononucleosis in children.

BACKGROUND: Although anti-viral capsid antigen (VCA)-immunoglobin M (IgM) is the most reliable serological marker of primary Epstein-Barr virus (EBV) infection, it could only be detected in limited cases of infectious mononucleosis in children. We analyzed anti-EBV antibodies by an enzyme-linked immunosorbent assay (ELISA), a sensitive method for detecting IgM antibody and compared these results with those obtained by a conventional indirect immunofluorescence (IF) method. METHODS: Anti-Epstein-Barr virus early antigen (EA)-IgM and nuclear antigen 1 (EBNA1)-IgG were examined by an ELISA assay in 180 sera from 70 infants and children with infectious mononucleosis, diagnosed serologically by standard IF methods. RESULTS: Although by IF, VCA-IgM was detected in only 37 of 70 (52.9%) of the sera from the acute phase of the disease, by ELISA, EA-IgM was detected in 65/70 (92.9%) of these sera. Among infants less than 12 months of age. EA-IgM was positive in 11/13 cases (84.6%) while VCA-IgM was detected in only 3/13 cases (23.1%). Anti-Epstein-Barr virus nuclear antigen 1-IgG was not detected by ELISA in the sera from the acute phase of infectious mononucleosis. Anti-EBNA was not detected by IF in about one-third of the sera during 6-8 months after onset of the disease, whereas by ELISA, EBNA1-IgG was detected in 93.0%. Sera that were positive or negative for both EA-IgM and EBNA1-IgG by ELISA were observed in several cases after the patients recovered from the disease. CONCLUSIONS: Although serodiagnosis by the combination of ELISA for EA-IgM and EBNAI-IgG was more sensitive than IF methods, especially in the case of infants and young children, several patients during convalescence and recovery might be judged as seronegative or as being in highly reactivated states.

Different effect of diastereoisomers of L-cystathionine sulfoxide on the stimulus coupled responses of human neutrophils.

The priming effect of L-cystathionine sulfoxide, which is one of the unusual cystathionine metabolites found in the urine of patients with cystathioninuria, on the stimulus-induced superoxide generation by human neutrophils was examined. The synthetic L-cystathionine sulfoxide significantly enhanced the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine [fMLP], opsonized zymosan [OZ], arachidonic acid [AA], and phorbol 12-myristate 13-acetate [PMA]. Then the synthetic L-cystathionine sulfoxide was separated into two diastereoisomers, CS-I and CS-II, which showed a peak at 76 and 83 min on chromatogram by amino acid analyzer, respectively. CS-I enhanced the superoxide generations induced by AA and PMA but not those induced by fMLP and OZ. On the contrary, CS-II enhanced the superoxide generations induced by fMLP and OZ but not those induced by AA and PMA. The superoxide generation induced by PMA with CS-I was suppressed by H-7 and was enhanced by genistein, while that by fMLP with CS-II was suppressed by genistein and was enhanced by H-7.

Soluble thrombomodulin and antibodies to bovine glomerular endothelial cells in patients with Henoch-Schönlein purpura.

AIM: To evaluate the clinical significance of soluble thrombomodulin and antiendothelial cell antibodies (AECA) in children with Henoch-Schönlein purpura. METHODS: Binding of serum AECA to bovine glomerular endothelial cells was evaluated by enzyme linked immunosorbent assay, cytotoxicity against glomerular endothelial cells by spectrophotometric assay, and soluble thrombomodulin concentrations by sandwich enzyme immunoassay. RESULTS: IgA AECA were detected in seven of 15 patients with Henoch-Schönlein purpura and nephritis, but were not detected in patients without nephritis or in controls. Patients with Henoch-Schönlein nephritis had raised titres of IgA AECA and serum thrombomodulin; severe proteinuria and renal histological changes were associated with raised titres of IgA AECA and raised serum thrombomodulin. No subjects had complement dependent cytotoxicity against glomerular endothelial cells. CONCLUSIONS: High titres of IgA AECA and raised serum thrombomodulin may be clinically useful markers of renal involvement in patients with Henoch-Schönlein purpura.